As is known, various diseases or injuries to the body give rise to the generation of protein fractions, e.g., isoenzymes, in blood which, if detected, can identify the particular injury or disease. For example, after a myocardial infarction, the enzyme creatine phosphokinase (CPK) is released into the blood where its activity can be measured by its ability to catalyze a specific chemical reaction. CPK has been considered to be the most reliable enzyme indicator of a myocardial infarction. The CPK activity rises in plasma about 2 to 4 hours after the infarction and persists for a period no greater than about 3 `days. The enzyme lactate dehydrogenase (LDH) is also an indication of a myocardial infarction; however, this does not increase above normal until about 1 or 2 days after the infarction and reaches a maximum at about the third or fourth day, whereupon it gradually decreases until it recedes back to normal in about 10 days.
Simply determining the total rise of all fractions of CPK or LDH enzymes in the blood serum is not an accurate method of diagnosis. For example, total CPK elevations can occur due to noncardiac conditions such as chronic alcoholism, cardioversion, cerebrovascular diseases, hypothyroidism, intramuscular injections and surgical trauma. A total rise in LDH can occur from either liver damage (e.g., hepatitis), a myocardial infarction, or muscle damage (e.g., myositis).
Both the CPK and LDH enzymes, as well as other enzymes, contain various fractions or isoenzymes of different net electrical charge. A rise in specific ones of these isoenzymes will, in contrast to a total rise in LDH or CPK, indicate a myocardial infarction or other diseased organ. The CPK enzyme contains the fractions or isoenzyme types MM, MB and BB. A rise in the MM and MB isoenzymes indicates a myocardial infarction; a rise in the MM isoenzyme indicates muscle damage only; whereas a rise in the BB-type only indicates brain damage. Similarly, LDH is composed of five isoenzymes (LD-1, LD-2, LD-3, LD-4 and LD-5), all of which appear in varying amounts dependent upon the type of tissue damage. Patient sera containing elevated amounts of LD-1 and LD-2 indicate a myocardial infarct; while elevated fractions of LD-3, LD-4 and LD-5 indicate damaged liver or muscle tissue.
The chief aim of past work has been to separate isoenzymes of CPK and LDH, as well as isoenzymes of other enzymes present in blood serum, by conventional electrophoretic techniques. However, routine use of the electrophoretic technique is limited since laborious gel preparations and poor staining techniques remain an integral part of the electrophoretic procedure.
Attempts have also been made to separate various isoenzymes with the use of ion exchange chromatography. Such attempts are described, for example, in an article by Ellen Schmidt, appearing in Clinica Chimica Acta, 15 (1967), pages 337-342. Sodium phosphate is used as a buffer in the ion exchange chromatographic technique described in the aforesaid article; and this requires dialysis of the fresh sera for at least six hours against a 0.008 molar potassium phosphate solution. Furthermore, the chromatographic technique described in that article takes at least 2 to 4 hours. As a result, while the method described in that article may be satisfactory for experimental studies, it is not satisfactory for rapid, routine use in a clinical laboratory by relatively unskilled technicians.
Takahashi et al, Clin. Chimica, Acta 38, 285-290 (1972) disclose a five-step process utilizing ion exchange chromatography to separate isoenzymes. The Takahashi et al process requires about 8 to 9 hours to complete since it depends upon dialysis of the sample to equilibrate it to the column used. Takahashi et al use a DEAE-Sephadex column and elute the sample by gradient elution. The Takahashi et al process is time consuming and causes difficulty in obtaining satisfactory results.
Richterich et al, Clinica Chimica Acta, 8, 178-192 (1963) disclose a batch elution technique wherein undialyzed serum is mixed with DEAE-Sephadex in a test tube and centrifuged. This process is time consuming and difficult to carry out since it requires repeated centrifugation steps and is not satisfactory for rapid, routine use in a clinical laboratory.